The receptor activator of NF-?B (RANK) ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily, plays an essential role in osteoclast (OC) formation, function and survival by binding to its receptor RANK, which is expressed on OC precursors and mature OC. Moreover, the RANKL/RANK system is also implicated in the pathogenesis of various bone disorders. Our long-term goal is to fully delineate RANK signaling in OC biology with an aim of identifying novel therapeutic targets for bone diseases. As a member of the TNF receptor (TNFR) family, RANK activates various signaling pathways including NF-?B, JNK, ERK, p38, NFATc1 and AKT to promote OC formation, function and/or survival by recruiting TNF receptor associated factors (TRAFs) via three specific motifs in its cytoplasmic domain (PFQEP369-373: Motif 1; PVQEET559-564: Motif 2 and PVQEQG604-609: Motif 3). Previously, we identified a TRAF-independent motif (IVVY535-538) in the RANK cytoplasmic domain that plays an essential role in OC formation by committing macrophages to the OC lineage in vitro. We have recently documented the importance of the IVVY motif in vivo and also identified the Ring1 and YY1 binding protein (RYBP) as a key molecule which interacts with the IVVY motif to regulate OC formation in vitro. Furthermore we have demonstrated that the role of the IVVY motif in OC formation requires the presence of a functional Motif 2 and/or Motif 3 in the RANK cytoplasmic domain, highlighting an important functional crosstalk between the IVVY motif-activated pathway and the TRAF-dependent signaling pathways. These new findings indicate that RANK signaling in OCs is more complex than previously thought and novel mechanisms are involved in OC formation. Thus, more work is needed to fully delineate RANK signaling in OC biology. The objectives of the current application are to elucidate the molecular mechanism by which RYBP regulates OC formation and to validate the role of RYBP in OC formation in vivo. Our central hypothesis is that 1) the IVVY motif mediates OC formation by recruiting RYBP, which in turn interacts with components of the polycomb (PcG) complexes to modulate gene expression in the early stage of OC differentiation; and 2) that efficient activation of the IVVY/RYBP-mediated signaling requires a functional crosstalk with TRAF proteins bound at Motif 2 and Motif 3. This hypothesis has been formulated based on our new findings. We propose to accomplish the objectives of the current application by pursuing the following three Specific Aims: 1) Delineate the molecular mechanism by which RYBP regulates OC formation; 2) Investigate the molecular mechanism of the dependence of the IVVY motif function on the two TRAF-binding sites; 3) Investigate the role of RYBP in OC formation in vivo. These studies will provide more crucial insights into RANK signaling in OC formation. Moreover, the RANKL/RANK system is also implicated in the pathogenesis of various bone diseases such as postmenopausal osteoporosis, bone loss in rheumatoid arthritis, and tumor-induced osteolysis, a full elucidation of RANK signaling may guide the development of new therapeutic targets for these bone diseases. PUBLIC HEALTH RELEVANCE: The proposed research seeks to investigate the molecular mechanism of the formation of osteoclasts, the bone-resorbing cells involved in various bone diseases including postmenopausal osteoporosis, inflammatory bone loss, and tumor-induced osteolysis. Thus, the proposed research is relevant to public health because a full elucidation of the molecular mechanism of osteoclast formation may reveal novel therapeutic targets for preventing and treating these bone diseases.